Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap-freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm(2) of membrane was assessed by conventional microscopy (CM) and computer-assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r(2) = 0.91 and CASA: r(2) = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm-egg-binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Semen analysis of Golden Retriever healthy dogs and those affected by muscular dystrophy.

This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers (n = 15) and from muscular dystrophy affected dogs (n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds.

α-6 integrin expression in bovine spermatogonial cells purified by discontinuous Percoll density gradient.

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.

Vitrification with glutamine improves maturation rate of vitrified / warmed immature bovine oocytes.

The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 µg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. L-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

Detection of protamine 2 in bovine spermatozoa and testicles.

BACKGROUND: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. OBJECTIVES: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. MATERIALS AND METHODS: For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. RESULTS: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. CONCLUSION: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Pharmacodynamic comparison of linezolid, teicoplanin and vancomycin against clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci collected from hospitals in Brazil.

Pharmacodynamic exposures, measured as the ratio of steady-state total drug area under the curve to MIC (AUC/MIC), were modelled using a 5000-patient Monte-Carlo simulation against 119 non-duplicate clinical isolates of Staphylococcus aureus and 82 coagulase-negative staphylococci (CNS) collected from hospitals in Brazil between 2003 and 2005. Pharmacodynamic targets included an AUC/MIC >82.9 for linezolid and >345 for teicoplanin and vancomycin, as well as a free drug AUC/MIC >180 for vancomycin. The cumulative fractions of response (CFRs) against all S. aureus isolates were 96.0%, 30.1%, 71.6%, 48.0% and 65.1% for linezolid 600 mg every 12 h, teicoplanin 400 mg every 24 h and 800 mg every 24 h, and vancomycin 1000 mg every 12 h and every 8 h, respectively. Using a free drug target for vancomycin improved the CFR to 94.6% for the high-dose regimen, but did not substantially alter results for the lower dose. CFRs against all CNS isolates were 97.8%, 13.4%, 34.6%, 10.9% and 31.3%, respectively, for the same antibiotic regimens. The CFR was reduced for all compounds among the methicillin-resistant isolates, except for linezolid against methicillin-resistant CNS. Sensitivity analyses did not alter the final order of pharmacodynamic potency against these isolates. Although higher doses of vancomycin and teicoplanin increased the CFR, the likelihood of achieving bactericidal targets was still lower than with linezolid. The results for the high-dose vancomycin regimen were highly dependent on the pharmacodynamic target utilised. These data suggest that linezolid has a greater probability of attaining its requisite pharmacodynamic target than teicoplanin and vancomycin against these staphylococci.

Effect of bovine sperm chromatin integrity evaluated using three different methods on in vitro fertility.

In vitro fertility potential of individual bulls is still relatively uncharacterized. Classical sperm analysis does not include the evaluation of all sperm characteristics and thus, some cell compartments could be neglected. In humans, sperm DNA integrity has already proven to have major influence in embryo development and assisted reproduction techniques successfully. In bovine, some studies already correlated chromatin integrity with field fertility. However, none of those have attempted to relate DNA assessment approaches such as chromatin deficiency (CMA3), chromatin stability (SCSA; AO+) and DNA fragmentation (COMET assay) to predict in vitro bull fertility. To this purpose, we selected bulls with high and low in vitro fertility (n = 6/group), based on embryo development rate (blastocyst/cleavage rate). We then performed CMA3, SCSA test and COMET assay to verify if the difference of in vitro fertility may be related to DNA alterations evaluated by these assays. For the three tests performed, our results showed only differences in the percentage of cells with chromatin deficiency (CMA3+; high: 0.19 ± 0.03 vs low: 0.04 ± 0.04; p = 0.03). No difference for chromatin stability and any of COMET assay categories (grade I to grade IV) was observed between high and low in vitro fertility bulls. A positive correlation between AO + cells and grade IV cells was found. Despite the difference between groups in CMA3 analysis, our results suggest that protamine deficiency in bovine spermatozoa may not have a strong biological impact to explain the difference of in vitro fertility between the bulls used in this study.

Morphological changes in mouse embryos cryopreserved by different techniques.

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.

Bone mineral density and inflammatory bowel disease severity.

Inflammatory bowel disease (IBD) is associated with low bone mineral density (BMD). In this study, the association between disease severity and BMD in patients with IBD was evaluated. Associations between BMD and the Montreal classification, disease activity and drug therapy were also tested. A cross-sectional prevalence study with a comparison group was conducted. One hundred and twenty-eight patients were evaluated: 68 patients with ulcerative colitis (UC), and 60 with Crohn's disease (CD). The control group consisted of 67 healthy subjects. All patients and controls had BMD measured and in IBD patients, current medications, hospitalization, and disease location, extent and phenotype, according to the Montreal classification, were recorded. Multiple correspondence analysis was applied to evaluate categorical variables. In the CD group, most patients were diagnosed between 17-40 years of age. Ileocolonic and non-stricturing non-penetrating disease were the most frequent disease location and behavior, respectively. In UC patients, extensive colitis was the most frequent disease location. UC and CD patients were more likely to have osteopenia than controls (OR=14.93/OR=24.38, respectively). In the CD group, male patients, perianal disease, penetrating behavior and age at diagnosis >40 years were associated with low BMD. Taking azathioprine and infliximab also seemed to be associated with osteopenia. In the UC group, we observed an association between low BMD and male patients, left colitis, corticosteroid use and hospitalization. Disease activity was not associated with osteopenia or osteoporosis in CD and UC patients. Disease severity seems to be associated with osteopenia in IBD patients.

Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation.

BACKGROUND: In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry. RESULTS: There was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters. CONCLUSION: Based on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.

Environmental contamination by multidrug-resistant Acinetobacter baumannii in an intensive care unit.

Contamination in an intensive care unit caused by multidrug-resistant Acinetobacter baumannii complex (MRAB)-colonized patients was evaluated using environmental and patient cultures. MRAB occurred in 21% of patients' cultures, 2.1% of 513 areas surrounding MRAB-patients, and one of 372 common areas. No transmission to other patients occurred. Barrier precautions and ethanol disinfection may prevent dissemination.

An outbreak of multiresistant Acinetobacter baumanii in a university hospital in São Paulo, Brazil.

A case-control (46 cases, 23 controls) study was done to determine risk factors for an outbreak of a multiresistant Acinetobacter baumanii (only susceptible to colistin) in a university hospital. The use of antecedent antibacterials and intubation were independent risk factors. No common source was found. With control measures, the outbreak resolved gradually.

Epidemiologic investigation of an outbreak of coagulase-negative Staphylococcus primary bacteremia in a newborn intensive care unit.

Infections due to coagulase-negative Staphylococcus (CNS) are an ever-increasing nosocomial problem, particularly in the pediatric population. The authors describe a cluster of three primary bloodstream infections due to CNS in a newborn intensive care unit that occurred between November 23 and December 2, 1992. Two children died as a direct consequence of the bacteremia; at autopsy, one had a large bacteria-containing thrombus extending from the insertion site of a central catheter to the superior vena cava. The children were placed in isolation, and the nursing and medical staff were given topical nasal mupirocin. Plasmid analysis performed later disclosed three different blood isolates that also were different from any of the staff's nasal isolates. The authors concluded that molecular methods such as plasmid analysis are important tools in identifying true outbreaks and can prevent needless interventions, such as those during this cluster.

Nosocomial infections caused by multiresistant Pseudomonas aeruginosa.

A case-control study was done to evaluate factors associated with nosocomial infections by multiresistant Pseudomonas aeruginosa (MRPA). Results showed that MRPA was associated with the use of immunosuppressive and antimicrobial drugs. Five typing methods indicated that the MRPA infections were due to multiple strains rather than a single strain.

Evaluation of the in vitro activity of cefepime compared to other broad-spectrum cephalosporins against clinical isolates from eighteen Brazilian hospitals by using the Etest.

The in vitro activity of cefepime was compared to that of ceftazidime, ceftriaxone, and cefotaxime in a multicenter study involving 10 clinical microbiology laboratories and clinical isolates from 18 Brazilian hospitals from 7 cities (4 states). A total of 982 isolates consecutively collected between December 1995 and March 1996 were susceptibility tested by using Etest and following the NCCLS procedures for agar diffusion tests. The cefepime spectrum was broader than that of the other broad-spectrum cephalosporins against both Gram-negative rods and Gram-positive cocci. Cefepime was particularly more active against Enterobacter sp. (MIC90, 2 micrograms/ml), Serratia sp. (MIC90, 2 micrograms/ml) and oxacillin-susceptible Staphylococcus aureus (MIC90, 3 micrograms/ml). Against Pseudomonas aeruginosa, cefepime (MIC90, 16 micrograms/ml) was slightly more active than ceftazidime (MIC90, 32 micrograms/ml) and 8- to 16-fold more active than ceftriaxone of cefotaxime (MIC90, > 256 micrograms/ml). Our results show that nosocomial bacteria, especially Gram-negative rods, have a high rate of cephalosporin resistance in Brazil. However, part of these resistant bacteria remains susceptible to cefepime. The Etest was shown to be an excellent method for multicenter studies of the in vitro evaluation of new antimicrobial agents.

Electric showers as a control measure for Legionella spp. in a renal transplant unit in São Paulo, Brazil. Legionellosis Study Team.

After an outbreak of legionnaires' disease Legionella pneumophila serogroup 1 in a renal transplant unit in São Paulo, Brazil, periodic hyperchlorination and flushing of pipes were instituted as control measures. These were only partially effective as every two to five months water cultures turned positive or new cases of the disease occurred. In November 1993 the hot water was disconnected from the unit and small, plastic electric showers were installed in each bathroom. Over a period of 12 months water from showers and taps was cultured for Legionella spp. every two weeks. On only one occasion was a water culture positive for L. pneumophila from a sink tap. No water sample obtained from showers was positive during the study period. No cases of legionnaires' disease occurred. We considered the use of electric showers an inexpensive and effective method of controlling the problem of Legionella spp. in the water system of our renal transplant unit.

An outbreak of nosocomial Legionnaires' disease in a renal transplant unit in São Paulo, Brazil. Legionellosis Study Team.

From June 1989 to March 1990 there were eight cases of Legionnaires' disease caused by Legionella pneumophila serogroup 1 in a renal transplant unit. There were seven cases of pneumonia and one case of pleural effusion. A study was conducted to identify the source of the outbreak. Legionella anisa was cultured from tap water. Twenty-seven staff members of the unit were serologically tested and antibody titres were positive in two. The probable source of infection was the potable water system. Control measures were hyperchlorination and heating of the water, after which there were no further cases during 5 months' follow up. We believe this is the first reported Legionnaires' disease outbreak in Latin America.

In vitro activity of ampicillin-sulbactam against clinical multiresistant Acinetobacter baumannii isolates.

We evaluated the in vitro activity of ampicillin-sulbactam in comparison with that of broad-spectrum antimicrobial agents against Acinetobacter baumannii isolates. Two hundred and twelve clinical isolates collected between January 1993 and March 1995 from two tertiary hospitals located in São Paulo, Brazil were tested for susceptibility by the disk diffusion method against several broad-spectrum antimicrobial agents, including imipenem, ciprofloxacin, ceftazidime, aztreonam, amikacin, and polymyxin B. All strains were susceptible to polymyxin B. The second most active compound was the combination ampicillin-sulbactam (88% susceptibility). Only 79% of the isolates were susceptible to imipenem. Ciprofloxacin was active against 60 (28%) and amikacin against 34 (16%) isolates. Ceftazidime was the most active cephalosporin; however, only 9% of the isolates were susceptible to this compound. Both aztreonam and ampicillin alone showed very poor activity against this species (1% susceptibility). The prevalence of severe infections due to A. baumannii is increasing very rapidly in the tertiary hospitals of São Paulo and there are very few options for the treatment of these infections. Polymyxin B is invariably in vitro active against this species; however, this compound can cause severe side effects and is not commercially available for intravenous use in Brazil and in several other countries. Our results indicated that the combination ampicillin-sulbactam may be an alternative drug for the treatment of infections due to multiresistant A. baumannii; however, further studies are necessary to evaluate the clinical role of this compound for the treatment of severe infections.

Production of a cloned calf from a fetal fibroblast cell line.

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).